title: The long march: a sample preparation technique that enhances contig length and coverage by high-throughput short-read sequencing.
abstract: High-throughput short-read technologies have revolutionized DNA sequencing by drastically reducing the cost per base of sequencing information. Despite producing gigabases of sequence per run, these technologies still present obstacles in resequencing and de novo assembly applications due to biased or insufficient target sequence coverage. We present here a simple sample preparation method termed the "long march" that increases both contig lengths and target sequence coverage using high-throughput short-read technologies. By incorporating a Type IIS restriction enzyme recognition motif into the sequencing primer adapter, successive rounds of restriction enzyme cleavage and adapter ligation produce a set of nested sub-libraries from the initial amplicon library. Sequence reads from these sub-libraries are offset from each other with enough overlap to aid assembly and contig extension. We demonstrate the utility of the long march in resequencing of the Plasmodium falciparum transcriptome, where the number of genomic bases covered was increased by 39%, as well as in metagenomic analysis of a serum sample from a patient with hepatitis B virus (HBV)-related acute liver failure, where the number of HBV bases covered was increased by 42%. We also offer a theoretical optimization of the long march for de novo sequence assembly.
authors.affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Katherine
authors.initials: K
authors.lastname: Sorber
authors.forename: Charles
authors.initials: C
authors.lastname: Chiu
authors.forename: Dale
authors.initials: D
authors.lastname: Webster
authors.forename: Michelle
authors.initials: M
authors.lastname: Dimon
authors.forename: J Graham
authors.initials: JG
authors.lastname: Ruby
authors.forename: Armin
authors.initials: A
authors.lastname: Hekele
authors.forename: Joseph L
authors.initials: JL
authors.lastname: DeRisi
mesh_terms: D000818:Animals; D020451:Contig Mapping / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific; D006509:Hepatitis B / Q000821:virology; D006801:Humans; D017093:Liver Failure / Q000821:virology; D008722:Methods; D010963:Plasmodium falciparum / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards
chemical_list: D012333:RNA, Messenger; D015252:Deoxyribonucleases, Type II Site-Specific